The goal of this application is to identify plasmid DNA partitioning systems that are active during both vegetative growth and sporulation in two different species of Bacillus. New tools for elucidating the mechanisms underlying plasmid segregation will be developed using the model organism B. subtilis, and then applied to B. megaterium. Utilizing two different Bacillus strains to study this problem will allow us to take advantage of the unique strengths that each organism and its indigenous plasmids offer, while maintaining a focused goal of understanding how conserved elements of the microbial DNA segregation machinery functions during sporulation to ensure that every spore inherits at least one copy of the plasmid. Plasmid segregation during sporulation provides an attractive system for studying DNA segregation because: 1) both the plasmids and the sporulation process are not nonessential; 2) the nearly 10 fold smaller volume of the forespore compared to the mothercell demands an efficient plasmid partitioning system; 3) cell-specific transcription factors become active during sporulation, allowing the development of powerful genetic and cell biology assays for DNA segregation during sporulation; and 4) many Bacillus plasmids do not encode proteins related to either the ParA or ParM families of plasmid segregation proteins, suggesting that novel partitioning mechanisms are at work. Since a wide variety of virulence factors are encoded on plasmids in pathogenic strains of Bacillus, understanding how plasmids are transmitted from one generation to the next is important for understanding the evolution and spread these key virulence determinants. These studies will allow the development of better genetic tools and plasmid vectors useful for manipulating many Bacillus species. [unreadable] [unreadable] [unreadable]